Basidiomycetes, basidiomycetes extract composition, health foods, and immunopotentiators

ABSTRACT

Basidiomycetes which is a novel mushroom having an excellent immunopotentiating action, etc., a Basidiomycetes extract composition, and health foods and immunopotentiators using the Basidiomycetes extract composition are provided. 
     Basidiomycetes has no basidium forming potential. In particular, Basidiomycetes is Basidiomycetes-X FERM BP-10011. A Basidiomycetes extract composition is extracted from them with an extraction solvent including at least one solvent selected from water and a hydrophilic solvent.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No.10/554,908, filed Oct. 31, 2005 now U.S. Pat. No. 7,517,682 (nowallowed); which is a 371 of PCT/JP2004/006418, filed May 6, 2004; thedisclosures of each of which are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to Basidiomycetes which is a novel mushroom(mushroom and other fungi will be collectively referred to hereinafteras mushroom) and has properties such as an immunomodulating effect, aBasidiomycetes extract composition, and health foods andimmunopotentiators using the Basidiomycetes extract composition.

BACKGROUND ART

Mushrooms have been used frequently since olden days as food materialshaving unique flavors and odors. They have also been used as Chineseherbal medicines as having physiological function activating actions,such as enhancement of immunocompetence, antimicrobial activity, controlof biorhythm, and prevention of senescence, or as folk medicines forcertain types of diseases. Studies of pharmacological ingredientsconcerned with mushrooms are in progress, resulting in the discovery ofingredients showing antibacterial and antiviral actions, a cardiotonicaction, a hypoglycemic action, a cholesterol lowering action, anantithrombotic action, and an antihypertensive action.

Proposals have been made for compositions which are usable as medicines,health foods, etc. and which comprise a mixture of dry products orextracts of two or more mushrooms selected from edible mushrooms amongbasidiomycetes, especially, Lentinus edodes (Berk.) Sing., Pleurotusostreatus (Jacq. ex Fr.) Quel., Pholiota nameko (T. Ito) S. Ito et Imai,Grifola frondosa, Flammulina velutipes (Curt. ex Fr.) Sing., andHypsizigus marmoreus (see Japanese Patent Application Laid-Open No.1999-152230).

In recent years, Agaricus Blazei murill (hereinafter referred to asagaricus mushroom), Phellinus linteus (Berk. et Curt) Tehg (hereinafterreferred to as mesimacobu) and so on have attracted attention as havingan anticancer action.

For examples, proposals have been put forward for a method forhigh-yield cultivation of mushrooms of the genus Phellinus such asmesimacobu (see Japanese Patent Application Laid-Open No. 1999-262329),a method for culturing mesimacobu mycelia for obtaining large amounts ofmycelia of mesimacobu (see Japanese Patent Application Laid-Open No.2001-178448) and a method for efficiently extracting ingredientscontained in agaricus mushroom by use of ultrasonic waves (see JapanesePatent Application Laid-Open No. 2001-278805).

As described above, various mushrooms have drawn attention as having ananticancer action, etc. However, they are not decisively effective, andthe advent of mushrooms having a better effect is desired.

DISCLOSURE OF THE INVENTION

The present invention has been accomplished in the light of theabove-mentioned circumstances. It is an object of the present inventionto provide Basidiomycetes which is a novel mushroom having an excellentimmunopotentiating action, etc., a Basidiomycetes extract composition,and health foods and immunopotentiators using the Basidiomycetes extractcomposition.

A first aspect of the present invention, for attaining the above object,lies in Basidiomycetes characterized by having no basidium formingpotential.

A second aspect of the present invention lies in the Basidiomycetes ofthe first aspect, characterized in that the Basidiomycetes isBasidiomycetes-X FERM BP-10011.

A third aspect of the present invention lies in a Basidiomycetes extractcomposition characterized by being extracted from Basidiomycetes, whichhas no basidium forming potential, with an extraction solvent includingat least one solvent selected from water and a hydrophilic solvent.

A fourth aspect of the present invention lies in the Basidiomycetesextract composition of the third aspect, characterized in that theBasidiomycetes is Basidiomycetes-X FERM BP-10011.

A fifth aspect of the present invention lies in the Basidiomycetesextract composition of the third or fourth aspect, characterized in thatthe Basidiomycetes extract composition is obtained by heating andextraction.

A sixth aspect of the present invention lies in the Basidiomycetesextract composition of any one of the third to fifth aspects,characterized in that the Basidiomycetes extract composition is obtainedby pressurization and extraction.

A seventh aspect of the present invention lies in a health foodcharacterized by containing, as an active ingredient, a Basidiomycetesextract composition extracted from Basidiomycetes having no basidiumforming potential.

An eighth aspect of the present invention lies in the health food of theseventh aspect, characterized in that the Basidiomycetes isBasidiomycetes-X FERM BP-10011.

A ninth aspect of the present invention lies in the health food of theseventh or eighth aspect, characterized in that the health food is in aform selected from a drink form, a snack form, a concentrated extractform, a powder, granules, tablets, and capsules.

A tenth aspect of the present invention lies in an immunopotentiatorcharacterized by containing, as an active ingredient, a Basidiomycetesextract composition extracted from Basidiomycetes having no basidiumforming potential.

An eleventh aspect of the present invention lies in theimmunopotentiator of the tenth aspect, characterized in that theBasidiomycetes is Basidiomycetes-X FERM BP-10011.

A twelfth aspect of the present invention lies in edible Basidiomycetescomprising a hypha mass formed by culturing the Basidiomycetes of thefirst or second aspect.

Basidiomycetes-X of the present invention described above contains largeamounts of polysaccharides (β-D-glucan) and has high antioxidantactivity, OH radical elimination activity, and an immunomodulatingeffect. This organism is preferred when used in health foods andimmunopotentiators which can be expected to exhibit pharmacologicalefficacy, such as prevention of senescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the results of measurements in Test Example 1.

FIG. 2 is a view showing the results of measurements in Test Example 2.

FIG. 3 is a view showing the results of measurements in Test Example 3.

FIG. 4 is a schematic view showing the mode of administration in TestExample 4.

FIG. 5 is a view showing the results of measurements in Test Example 4.

BEST MODE FOR CARRYING OUT THE PRESENT INVENTION

Basidiomycetes, referred to in the present invention, is abasidiomycete, and has properties such that it has no basidium formingpotential, although beak-shaped processes (clamps) are observed. Inthese respects, this basidiomycete is distinguished from otherbasidiomycetes. That is, even when cultured, Basidiomycetes does notform basidia, and only forms sclerotia (hypha masses).

Such Basidiomycetes was obtained as a result of search formicroorganisms in the natural world. It was isolated, and deposited withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, Chuo Dai-6, Higashi 1-1-1, TsukubaCity, Ibaragi Prefecture, Japan 305-8566, as Basidiomycetes-X (AccessionNo.: FERM BP-100 μl), under the terms of the Budapest Treaty.

The organism according to the present invention forms no conidia,namely, has no asexual generation.

That is, when this organism is cultured on a potato glucose agar medium,cultured hyphae have clamps, and are smooth, but form no conidia, andform no fruit bodies. When the shape and color of the colony surface areobserved, a light pink hypha mass is formed in the colony. If aplurality of hypha masses are formed within the colony which has grownconcentrically from the site of inoculation, the hypha masses areinterconnected by mycelial strands. The color of the back of the colonyis light pink.

When this organism is cultured on a glucose-dry yeast agar medium,cultured hyphae have clamps, and are smooth, but form no conidia, andform no fruit bodies. When the shape and color of the colony surface areobserved, light pink to white hypha masses are formed in the colony.Hypha masses of 5 to 6 mm in thickness are formed, with the site ofinoculation as a center. The color of the back of the colony is lightpink to white.

The optimum growth conditions for the organism of the present inventionare, for example, pH 5.0 to 6.0 and a temperature of 22 to 26° C. Thegrowth range is, for example, pH 4.0 to 7.5 and a temperature of 5 to30° C.

Basidiomycetes, the organism according to the present invention, can becultured by an ordinary method, and the method of its culture is notlimited.

The Basidiomycetes extract composition of the present invention may beany cell contents extracted from hypha masses obtained by culturingBasidiomycetes, and the method of extraction is not limited. To extractcell contents from the hypha masses with high efficiency, it ispreferred to damage cell walls, for example, by freezing of the hyphamasses, if desired, thaw the frozen masses, and then crush them by meansof a mixer or the like, followed by extraction. The method of extractionis not limited, but extraction is performed at room temperature, orunder heating conditions, or under pressure, with the use of water, alower alcohol or the like, or an extraction solution furtherincorporating an acid, an alkali or other additive. Generally, thecrushed product is simmered in hot water for extraction, or the crushedproduct mixed with water or alcohol, or water incorporating an alkali isextracted under pressure, for example, of the order of 100 MPa to 700MPa, preferably, 300 MPa to 600 MPa.

An example of extraction in hot water will be described. For example,frozen Basidiomycetes-X hypha masses are thawed at room temperature, andcrushed using a mixer. The ratio of the crushed Basidiomycetes-X hyphamasses to water as an extraction solvent is set, for example, at 1:5.For example, 50 g of the crushed Basidiomycetes-X hypha masses areplaced in a glass bottle, 250 ml of water is added, and the glass bottleis covered with a lid. A towel is spread at the bottom of a pan, wateris poured over the towel, and the glass bottle filled with the crushedhypha masses is placed on the towel, followed by heating and boiling.After boiling, heating is continued for 90 minutes. After cooling, thesolids and liquid are separated to obtain a Basidiomycetes-X extract.The pH of the extract shows, for example, 6.3 to 6.5.

The resulting extract is concentrated, where necessary, to obtain aBasidiomycetes extract composition. Concentration of the extract is notlimited, but is performed, for example, in the following manner:

The resulting Basidiomycetes-X extract is transferred into a beaker, andheated and evaporated for concentration. At this time, the extract showsa light beige to brown color, and begins to bubble vigorously. However,evaporation and concentration are continued further, and concentrationis completed, for example, at a time when the concentrated extractbecomes tarry at pH 4.9 and a density of 1.25 g/cm³. The concentratedextract gives off a soy sauce-like odor. The yield of the concentratedextract from the Basidiomycetes-X hypha masses at this point in time isan average of 12%.

The thus obtained concentrated extract becomes very viscous as it cools.Thus, the concentrated extract needs to be transferred into a storagecontainer at the same time as the completion of concentration. Theconcentrated extract transferred into the storage container ispreferably cooled as it is, and then stored in a refrigerated or frozenstate.

The Basidiomycetes extract composition of the present invention can beused in health foods or medicines, such as immunopotentiators, in theform of, for example, drinks, snacks, concentrated extract, powder,granules, tablets, or capsules. The amount of the Basidiomycetes extractcomposition added may be set, as appropriate, in accordance with uses,and is not limited.

Furthermore, hypha masses obtained by culturing Basidiomycetes, which isthe organism according to the present invention, can be used for eatingpurposes, and are excellent in taste and organoleptic sensation.

The Basidiomycetes of the present invention, when cultured, forms hyphamasses in accordance with an environment where it is cultured. That is,when the Basidiomycetes is cultured in a vessel of a predeterminedshape, hypha masses of the shape of the vessel are obtained. Thus,edible Basidiomycetes easy to use for eating purposes is obtained. Theresulting edible Basidiomycetes may be used raw as a food material, ormay be used in a frozen or dried state as a food material, butpreferably, is used as a raw material or a frozen material.

In connection with the method of culture, the Basidiomycetes can becultured by an ordinary method as stated earlier, and the culture methodis not limited. For example, however, an agar medium, a sawdust medium,or a liquid medium, which has been supplemented with a suitable nutrientsource and sterilized, is aseptically inoculated with a cultured strainof the invented organism, or the seed organism, and the inoculum iscultured under appropriate temperature conditions, whereby hypha massesof Basidiomycetes-X can be obtained.

The method of cooking the edible Basidiomycetes used as a food materialis not limited. However, the edible Basidiomycetes can be cooked in thesame various ways as for ordinary mushrooms, such as simmering,pan-frying, roasting, and deep-frying, without any limitations. Sincethe edible Basidiomycetes gives an excellent organoleptic sensation anddoes not taste characteristically, it can be used widely in variousprepared foods.

Eating the edible Basidiomycetes is, needless to say, assumed to obtainthe same effect as when eating the Basidiomycetes extract composition.

EXAMPLES

The present invention will now be described more concretely withreference to the examples offered below. Examples 1 to 4 representcultivation examples of Basidiomycetes-X, and Examples 5 to 9 representextraction examples.

Example 1 Separation from Hypha Masses

(1) Preparation of Culture Media

PSA and PDA culture media were prepared in accordance with theformulations shown in Table 1. Each of the culture media was dispensedinto test tubes or Erlenmeyer flasks. Then, silicon caps (or cottonstoppers) were applied, and the stoppered containers were subjected tohigh pressure steam sterilization in an autoclave for 20 minutes at 121°C. Then, the test tubes were inclined while hot after sterilization toform slant media. On the other hand, the Erlenmeyer flasks were allowedto stand to form plate media.

TABLE 1 PSA culture medium PDA culture medium Extract of 200 g ofpotatoes Extract of 200 g of potatoes boiled for 20 minutes boiled for20 minutes 20 g sucrose 20 g glucose 15 g agar 15 g agar Total amount 1liter Total amount 1 liter(2) Separation from Hypha Masses

Larger Basidiomycetes-X hypha masses were broken manually, and sliceswere cut from Basidiomycetes-X sections with a scalpel which had beenflame sterilized and cooled. The PDA and PSA slant media of (1) wereeach inoculated with the Basidiomycetes-X slices using tweezers whichhad been flame sterilized and cooled. This procedure was performed underaseptic conditions within an aseptic box or a clean bench.

(3) Culture

The inoculum was cultured in an incubator at 24° C., and found togenerate the organism in 24 to 48 hours. After generation of theorganism, culture was continued at 24° C. Hyphae grew on the agar mediain 14 days.

Example 2 Culture on Sawdust Medium for Hypha Mass Production

(1) Culture of Seed Organism

Water was added to 1 liter of sawdust, 15 g of defatted bran, 15 g ofwheat bran, and 5 g of SANPEARL (hypha activator, Nippon PaperIndustries), and the mixture was vigorously stirred. This mixture forculture was adjusted such that when it was firmly gripped, water exuded(moisture content of the mixture: about 70%), whereby a sawdust mediumwas prepared. This culture medium was placed in an Erlenmeyer flask,which was covered with a silicon cap. Then, the Erlenmeyer flask wassubjected to high pressure steam sterilization in an autoclave for 40minutes at 121° C. Twenty-four hours after the sterilization,Basidiomycetes-X hyphae during culture on the slant media in Example 1were inoculated into the sawdust medium within an aseptic box by anaseptic operation. The inoculation was carried out such that no damagewas caused to the hyphae, with a sterilized triangular knife being usedto cut off a part of the slant medium. The density of the inoculationwas 20 to 30% of the surface area of the sawdust medium. When theinoculum was cultured at 24° C., the organism was generated in 3 days(in 5 days at the latest). After a lapse of 30 days, the sawdust mediumin the Erlenmeyer flask was full of the organism.

(3) Generation of Hypha Masses

A sawdust medium was prepared in the same manner as in (1). This culturemedium was placed in a polypropylene bottle, which was stoppered, andsubjected to high pressure steam sterilization in an autoclave for 40minutes at 121° C. Twenty-four hours after the sterilization, the seedorganism cultured in (1) was inoculated into the sawdust medium in thepolypropylene bottle by an aseptic operation within an aseptic box afteraseptic treatment. The density of the inoculation was such that thesurface area of the sawdust medium was nearly covered with the inoculum.When the inoculum was cultured at 24° C., the organism was generated in48 hours. After a lapse of 60 days, the entire sawdust medium within thepolypropylene bottle was full of hyphae. After a further lapse of 40 to50 days, hyphae spread on the inner wall of the polypropylene bottle,forming mycelial strands. When culture was continued further, hyphamasses were formed.

Example 3 Culture on Liquid Medium for Hypha Mass Production

Potatoes (200 g) cut to a size of 1 cm square were boiled using purifiedwater, followed by heating for 20 minutes. After cooling, the solids andthe liquid were separated, and distilled water was added to theresulting potato leachate and 20 g of sucrose to give a total amount of1 liter, thereby preparing a liquid medium. This liquid medium wasdispensed in an amount of 5 ml each into test tubes. The test tubes werecovered with silicon caps, and sterilized (high-pressure steamsterilization for 20 minute at 121° C. or atmospheric pressure steamsterilization for 8 hours at 100° C.). Then, the liquid media wereinoculated by an aseptic operation within an aseptic box after aseptictreatment such that the lower ends of slices of Basidiomycetes-X duringculture on the slant media in Example 1 contacted the liquid media. Whenthe inoculum was cultured at 24° C., the organism was generated in 48hours. Upon further culture, hypha masses were formed in contact withthe liquid media.

Example 4 Culture on Agar Medium for Hypha Mass Production

Potatoes (200 g) cut to a size of 1 cm square were boiled using purifiedwater, followed by heating for 20 minutes. After cooling, the solids andthe liquid were separated, and distilled water was added to theresulting potato leachate, 20 g of sucrose, and 1 g (0.1%) agar to givea total amount of 1 liter, thereby preparing an agar medium. Normally,to prepare an agar medium, 1.5 to 2.0% of agar (15 to 20 g based on 1liter of the resulting medium) is added, but 0.1% of agar was added tofacilitate separation of hypha masses after culture and the agar medium,and also to maintain the physical strength of the liquid medium becauseslices of Basidiomycetes-X tend to settle out in the liquid medium. This0.1% agar medium was dispensed in an amount of 5 ml each into testtubes. The test tubes were covered with silicon caps, and then subjectedto high pressure steam sterilization for 20 minute at 121° C. Then,slices were cut from Basidiomycetes-X hypha masses during culture on theslant media in Example 1, and inoculated into the 0.1% agar media by anaseptic operation within an aseptic box after aseptic treatment. Whenthe inoculum was cultured at 24° C., the organism was generated in 48hours. Upon further culture, hypha masses were formed.

Example 5 Production of Concentrated Basidiomycetes-X ExtractComposition by Decoction

To cause damage to the cell walls of the hyphae and facilitate theleaching-out of the cell contents, fresh Basidiomycetes-X hypha masseswere refrigerated or frozen. The frozen Basidiomycetes-X hypha masseswere thawed at room temperature, and crushed using a mixer. The crushedBasidiomycetes-X hypha masses (50 g) were placed in a glass bottle, 250ml of water was added, and the glass bottle was covered with a lid. Atowel was spread at the bottom of a pan, water was poured over thetowel, and the glass bottle filled with the crushed hypha masses wasplaced on the towel, followed by heating and boiling. After boiling,heating was continued for 90 minutes. After cooling, the solids andliquid were separated to obtain a Basidiomycetes-X extract composition.The pH of the extract was 6.3 to 6.5.

The resulting Basidiomycetes-X extract composition was transferred intoa beaker, and concentrated upon heating and evaporation. The extractcomposition showed a light beige to brown color, and began to bubblevigorously. However, evaporation and concentration were continuedfurther, and concentration was completed at a time when the concentratedextract composition became tarry at pH 4.9 and a density of 1.25 g/cm³.The concentrated Basidiomycetes-X extract composition gave off a soysauce-like odor. The yield of the concentrated Basidiomycetes-X extractcomposition from the Basidiomycetes-X hypha masses at this point in timewas an average of 12%. The Basidiomycetes-X extract composition becomesvery viscous as it cools. Thus, simultaneously with the completion ofconcentration, the concentrate was transferred into a storage containerand, after cooling, was stored as such in a refrigerated or frozenstate.

Example 6 Production of Basidiomycetes-X Extract Composition byDecoction

To cause damage to the cell walls of the hyphae and facilitate theleaching-out of the cell contents, fresh Basidiomycetes-X hypha masseswere refrigerated or frozen. Then, the frozen Basidiomycetes-X hyphamasses were thawed at room temperature.

The Basidiomycetes-X hypha masses (wet weight 20 g) after thawing wereweighed, cut to a size of 0.5 cm square, and placed in a beaker. After100 ml of water was added, the contents of the beaker were cooked gentlyat 90° C., and the solution was boiled down to a half of the originalamount. Then, water was added to restore the original amount. Themixture was filtered through a gauze to remove the solids. Then, thefiltrate was sealed up, and stored in a refrigerator for use as aBasidiomycetes-X extract composition of Example 6.

Example 7 Production of Basidiomycetes-X Extract Composition by HighPressure Treatment

The Basidiomycetes-X hypha masses (wet weight 20 g) treated in the samemanner as in Example 6 were taken into a vinyl bag, and 100 ml of waterwas added. Then, the vinyl bag was deaerated under reduced pressure, andsealed. The vinyl bag was set in an ultra-high pressure apparatus (aproduct of Kobe Steel; capable of treatment at 700 MPa), and treated for10 minutes at a hydrostatic pressure of 400 MPa. The treated mixture wasfiltered through a gauze, and the filtrate was stored in a refrigeratedstate for use as a Basidiomycetes-X extract composition of Example 7.

Example 8 Production of Basidiomycetes-X Extract Composition by HighPressure Treatment

A composition produced in the same manner as in Example 7, except fortreatment at a hydrostatic pressure of 600 MPa, was put to use as aBasidiomycetes-X extract composition of Example 8.

Example 9 Production of Basidiomycetes-X Extract Composition by HighPressure Treatment

A composition produced in the same manner as in Example 8, except forthe use of 100 ml of a 0.1% KCl aqueous solution instead of 100 ml ofwater, was put to use as a Basidiomycetes-X extract composition ofExample 9.

Test Example 1 Measurement of Active Oxygen (Hydroxy Radicals)Elimination Activity

The activity of eliminating hydroxy radicals was measured by ESR(electron spin resonance) using H₂O₂/UV as a hydroxy radical generationsource, and dimethylpyrroline-N-oxide (DMPO) as a spin trapper.

DMPO (40 mM) and 20 mM of hydrogen peroxide were added to a constantamount of the Basidiomycetes-X extract composition in each of Examples 6to 9, and purified water was added to give a total amount of 300 μl. Themixture was irradiated with UV (band width 20 nm) at a wavelength of 245nm, and the resulting hydroxy radical addition product of DMPO wasobserved for ESR signals. Based on changes in the intensity of thesignals, the hydroxy radical elimination activity of the extractcomposition was determined. The results are shown in FIG. 1.

As shown in FIG. 1, the larger the amount of the Basidiomycetes-Xextract composition, the higher the elimination rate of the hydroxyradicals became. Example 7, which involved extraction by high pressuretreatment at 400 MPa in a water solvent, obtained the highest hydroxyradical elimination rate.

Test Example 2 Measurement of Active Oxygen (Superoxide Anion Radicals)Elimination Activity

The activity of eliminating superoxide anion radicals was measured byESR (electron spin resonance) in accordance with the spin-trap methodusing a xanthine-xanthine oxidase system as a superoxide anion radicalgeneration system, and DMPO as a spin trapper.

DMPO (0.3 mM), 0.5 mM of hypoxanthine, and 1 mM ofdiethylenetriaminepentaacetic acid (DTPA) were added to a constantamount of the Basidiomycetes-X extract composition in each of Examples 6to 9, and 0.2M PBS was added to give a total amount of 300 μl. Xanthineoxidase was added in a concentration of 0.1 unit/ml, and the resultingDMPO-OOH (superoxide anion radical addition product of DMPO) wasobserved for ESR signals. Based on changes in the intensity of thesignals, the elimination activity of the extract composition wasdetermined. The results are shown in FIG. 2.

As shown in FIG. 2, the larger the amount of the extract composition,the higher the elimination rate of the superoxide anion radicals became.Example 7, which involved extraction by high-pressure treatment at 400MPa in a water solvent, obtained the highest superoxide anion radicalelimination rate. These results were similar to those in Test Example 1.

Test Example 3 Measurement of Active Oxygen (Hydroxy Radicals)Elimination Activity

The activity of eliminating hydroxy radicals was measured by the ESRspin-trap method using Fenton reaction as a hydroxy radical generationsource, and DMPO as a spin trapper.

Dimethylpyrroline N-oxide (DMPO) (20 mM), 10 mM of hydrogen peroxide,and 0.1 mM of FeSO₄ were added to a constant amount (10 or 20 μl) eachof an extract obtained by decocting and extracting dried agaricus (aproduct of Truffle Japan) under the same conditions as in Example 6, anextract obtained by decocting and extracting dried reishi mushroom(Ganoderma lucidum (Leyss. ex Fr.) Karst.; a product of Truffle Japan)under the same conditions as in Example 6, and the Basidiomycetes-Xextract composition in each of Examples 6 to 9. Purified water wasfurther added to give a total amount of 300 μl, and the resultingmixture was used as an assay sample. Based on changes in the intensityof the signals of DMPO-OH (a hydroxy radical addition product of DMPO)one minute after addition of FeSO₄, the elimination activity wasdetermined. The results are shown in FIG. 3.

As shown in FIG. 3, Example 6, which involved extraction by decoction,was not successful in estimating detailed elimination activity, becausethe extract of Example 6, when at a low concentration, interacted withiron ions as did the reishi mushroom extract, and caused increases in,rather than the elimination of, DMPO-OH signals. When the extract ofExample 6 was used at a high concentration minimal in influence on thesignals, and was compared with the other extracts, the extract ofExample 6 showed comparable elimination activity to that of the agaricusextract. Examples 7 to 9 involving extraction by high pressure treatmentgave higher elimination activity than did agaricus and reishi mushroom.

Test Example 4 Measurement of Immunomodulating Effect

Mice used were C3H/HeJ mice of CLEA Japan. C3H/HeJ mice showdeteriorated immunity when elderly. In the present study, “retirees” (20to 30 week old) were used as elderly mice. The concentratedBasidiomycetes-X extract composition of Example 5 was used asBasidiomycetes-X. For this assay, Associate Professor Akira Yanagawa,Applied Pharmacology Lab., 3rd Dept., Institute of Medical Science, St.Marianna Univ. School of Medicine cooperated, and performed work unpaid.

The retiree mice were divided into groups of 10 mice, and allocated to aBasidiomycetes-X treatment group administered the concentratedBasidiomycetes-X extract composition in a dose of 0.2 ml once daily, anda control group receiving 0.2 ml physiological saline once daily. TheBasidiomycetes-X or physiological saline was administered orally for 14consecutive days using a stomach tube.

Ten days after initiation of the treatment, 0.1 ml of 10% sheep redblood cells (SRBC) diluted with phosphate buffered physiological saline(PBS) (i.e., cell count 2×10⁸) was administered intraperitoneally to themice. After a lapse of 4.5 days, mouse splenic cells were removed, andthe number of plaque forming cells (PFC) was counted by the method ofJerne. The PFC count was compared with that in the control group. Theresults of measurement of the PFC count in the control group are shownin Table 2, and the results of measurement of the PFC count in theBasidiomycetes-X treatment group are shown in Table 3. A schematic viewof the modes of administration of Basidiomycetes-X or physiologicalsaline, and SRBC is shown in FIG. 4. The results of assay are shown inFIG. 5.

TABLE 2 Cell count Cell in cell suspension suspension seeded PFC (×10⁶into Petri (/Petri PFC (/10⁶ cells) No. cells/ml) dish (ml) dish)Individual Mean SD 1 31 0.2 0 0 2 51.25 0.03 10.33 6.72 3 59.75 0.03198.98 110.98 4 53.65 0.03 150.68 93.59 5 76.15 0.03 162.54 71.13 75.39962.98075 6 42 0.2 0 0 7 62.65 0.03 148.76 79.13 8 26.25 0.03 126.36160.36 9 38.25 0.03 203.42 177.12 10 63.45 0.03 104.64 54.96 *PFC/Petridish ÷ (cell suspension seeded into Petri dish × cell count in cellsuspension) = PFC individual

TABLE 3 Cell count Cell in cell suspension suspension seeded PFC (×10⁶into Petri (/Petri PFC (/10⁶ cells) No. cells/ml) dish (ml) dish)Individual Mean SD 1 20 0.03 263.33 438.88 2 30.75 0.2 8078.4 1077.12 355.25 0.2 4125.6 373.36 4 41.5 0.03 483 387.95 5 32 0.03 565.33 588.891345.9 1495.324 6 43.5 0.03 826.34 633.21 7 62 0.2 6236.42 502.9 8 53.450.03 7326.6 4567.77 9 36.5 0.2 9236.6 1265.53 10 79.75 0.03 8672 3626.39

As shown in Tables 2 and 3, the Basidiomycetes-X treatment group showedthe PFV value of 1345.9 and the SD value of 1495.324, which were about20 times those in the control group showing the PFC mean value of 75.399and the SD value of 62.98075. Two-sided test according to equaldistribution in Student T test showed Basidiomycetes-X to increase thePFC count significantly at P=0.023363 (p<0.05).

The experiments of the present study demonstrated the concentratedBasidiomycetes-X extract composition to increase the PFC countsignificantly in comparison with the physiological saline in the controlgroup. The PFC experimental method using C3H/HeJ mice is the methodcommonly practiced as a standard screening method for testing theimmunomodulating potential. The increase in the PFC count in the agedmice showed that the concentrated Basidiomycetes-X extract compositionenhances compromised immunocompetence.

Test Example 5 Course of Immunocompetence Parameters in Cancer Patients

The course of immunocompetence parameters in cancer patients (case 1 tocase 6) was monitored during treatment with the concentratedBasidiomycetes-X extract composition of Example 5 to investigate theimmunopotentiating effect of the concentrated Basidiomycetes-X extractcomposition. For this study, Associate Professor Akira Yanagawa, AppliedPharmacology Lab., 3rd Dept., Institute of Medical Science, St. MariannaUniv. School of Medicine, cooperated, and performed work unpaid.

Concretely, 1 ml of purified water was added to 1 ml of the concentratedBasidiomycetes-X extract composition, and the mixture was orallyadministered 3 times daily, after each meal. This treatment lasted for 3weeks.

As immunocompetence parameters, BML (BML, Inc.) was asked to measure thefollowing items before and after treatment on a blind basis. The resultsare shown in Tables 4 to 15. The six patients with cancer were alldifferent in the primary lesion of cancer. Since it bears no meaning tocalculate the mean value of these six patients, the values of theindividual patients were enumerated.

As NK cells:

Two color (as activity evaluation of NK cells)

CD57+CD16+(%) NK activity moderate

CD57+CD16−(%) NK activity weak

CD57−CD16+(%) NK activity strong

CD57−CD16−(%)

As total activated NK cell count:

CD3+HLA−DR+(%) Activated CD3 cells

Besides, the leukocyte count, the lymphocytes (%) and lymphocyte countin the leukocyte differential count were also measured. Furthermore, thecooperative patients were requested to enter in diaries changes insymptoms during treatment.

(Case 1) In July 2000, total sigmoidectomy was performed for sigmoidcolon cancer. In 2002, recurrent carcinoma was confirmed duringoperation for parietal cicatricial hernia. Then, ileus frequentlyoccurred.

TABLE 4 NK cell system Two color (activity evaluation of NK cells)Before treatment After treatment (lymphocyte count: LC) (LC) CD57+CD16+(%) NK 6.6% (149) 8.0% (250) activity moderate CD57+CD16− (%) NK 17.6%(396) 17.6% (549) activity weak CD57−CD16+ (%) NK 4.6% (104) 4.1% (128)activity strong CD57−CD16− (%) 71.2% 70.3%

TABLE 5 As total activated NK cell count Before treatment Aftertreatment (lymphocyte count: LC) (LC) CD3+HLA−DR+ (%) 9.9% (223) 10.6%(331) Activated CD3 cells

In case 1, the lymphocytes having moderate and weak NK cell activitywere markedly increased as compared with the pretreatment levels. TheCD57−CD16+ cells having strong NK activity showed the post-treatmentvalue of 4.1%, apparently indicating a decrease in %. However, theactual count of lymphocytes increased from 104 to 128. In regard to theCD3+HLA−DR+cells as an object of assay for the entire profile of NKcells, the post-treatment value was 10.6% (331), showing an increaseover the pretreatment value of 9.9% (223).

(Case 2) Total resection of left breast cancer was performed in October1999. Then, the carcinoma relapsed, and has currently metastasized tothe lung, bone, brain, and meninx. Even after radiotherapy for meningealdissemination, cranial nerve paralysis made the patient bedridden.Spinal cord metastasis also caused progressive right upper limbparalysis. The systemic condition is severely poor for terminal cancer.

TABLE 6 NK cell system Two color (as activity evaluation of NK cells)Before treatment After treatment (lymphocyte count: LC) (LC) CD57+CD16+(%) NK 12.6% (136) 9.4% (111) activity moderate CD57+CD16− (%) NK 5.76%(62) 6.8% (80) activity weak CD57−CD16+ (%) NK 9.4% (102) 7.0% (83)activity strong CD57−CD16− (%) 72.3% 76.8%

TABLE 7 As total activated NK cell count Before treatment Aftertreatment (lymphocyte count: LC) (LC) CD3+HLA−DR+ (%) 5.7% (62) 3.6%(43) Activated CD3 cells

In case 2, the influence of the concentrated Basidiomycetes-X extractcomposition on NK cells was not observed.

(Case 3) In August 2001, mucinous cystadenocarcinoma and bilateralmetastatic ovarian tumor necessitated resection. Then, carcinomatousperitoneal dissemination and carcinomatous inflammation resulted inlarge amounts of ascitic retention. Currently, the patient is bedriddenbecause of terminal cancer.

TABLE 8 NK cell system Two color (as activity evaluation of NK cells)Before treatment After treatment (lymphocyte count: LC) (LC) CD57+CD16+(%) NK 5.5% 5.8% activity moderate CD57+CD16− (%) NK 21.8% 16.0%activity weak CD57−CD16+ (%) NK 7.7% 6.8% activity strong CD57−CD16− (%)65.0% 71.4%

TABLE 9 As total activated NK cell count Before treatment Aftertreatment (lymphocyte count: LC) (LC) CD3+HLA−DR+ (%) 15.5% 13.1%Activated CD3 cells

In Case 3, a slight increase in CD57+CD16+ lymphocytes having moderateNK activity was observed.

(Case 4) In 2001, chemotherapy and radiotherapy were performed forpulmonary carcinoma (squamous cell carcinoma, T2N3M0). Then, anoperation for total resection of the left lung was performed. In 2002,metastatic brain tumor (cerebral metastasis of lung cancer) necessitatedmetastatic brain tumor resection. However, multiple cerebral metastasisoccurred as a complication in the same year.

TABLE 10 NK cell system Two color (as activity evaluation of NK cells)Before treatment After treatment (lymphocyte count: LC) (LC) CD57+CD16+(%) NK 5.3% 2.6% activity moderate CD57+CD16− (%) NK 1.1% 0.6% activityweak CD57−CD16+ (%) NK 8.8% 5.9% activity strong CD57−CD16− (%) 84.8%90.9%

TABLE 11 As total activated NK cell count Before treatment Aftertreatment (lymphocyte count: LC) (LC) CD3+HLA−DR+ (%) 5.2% 4.7%Activated CD3 cells

In this patient, the NK cell increasing effect of the concentratedBasidiomycetes-X extract composition was not observed.

(Case 5) In October 2001, lung cancer (adenocarcinoma) was noted. Atdiagnosis, metastasis to the right cervical lymph node was observed, andmetastasis to the hilar lymph nodes was complicated by superior venacava syndrome. Therapies included 60 Gy radiation of the right neckregions and chemotherapy (CBDCA+TAy 4 courses). The superior vena cavaremained completely obstructed, and carcinomatous pleuritisconcomitantly occurred. The medications were frequently given in thepulmonary cavity, but decreased the lesion only mildly. In addition,metastasis to the brain was recently confirmed upon CT.

TABLE 12 NK cell system Two color (as activity evaluation of NK cells)Before treatment After-treatment (lymphocyte count: LC) (LC) CD57+CD16+(%) NK 12.1% (256) 17.5% (431) activity moderate CD57+CD16− (%) NK 36.5%(773) 44.6% (1099) activity weak CD57−CD16+ (%) NK 4.3% (91) 6.0% (148)activity strong CD57−CD16− (%) 47.0% 31.9%

TABLE 13 As total activated NK cell count Before treatment Aftertreatment (lymphocyte count: LC) (LC) CD3+HLA−DR+ (%) 22.2% (469) 32.3%(796) Activated CD3 cells

In the present case, all the NK cell parameters were increased, and theoral administration of the concentrated Basidiomycetes-X extractcomposition increased NK cell activity and the number of NK cells. Thiscase is evaluated as a case of excellent response.

(Case 6) In July 2002, gastric cancer (Borrmann type I gastric carcinomain the gastric vestibule) was found. However, the patient did not wishfor an operation, and fell into the state of terminal cancer.

TABLE 14 NK cell system Two color (as activity evaluation of NK cells)Before treatment After treatment (lymphocyte count: LC) (LC) CD57+CD16+(%) NK 30.1% 30.9% activity moderate CD57+CD16− (%) NK 7.8% 7.1%activity weak CD57−CD16+ (%) NK 6.2% 6.7% activity strong CD57−CD16− (%)55.9% 55.3%

TABLE 15 As total activated NK cell count Before treatment Aftertreatment (lymphocyte count: LC) (LC) CD3+HLA−DR+ (%) 9.9% 9.1%Activated CD3 cells

In the present case, CD57−CD16+(%) (NK activity strong) andCD57+CD16+(%) (NK activity moderate) were increased.

Even among terminal cancer patients., two types are present, patientswho can still live the usual daily life, and bedridden patients in theterminal stage. The concentrated Basidiomycetes-X extract composition,when ingested, was expected to obtain a marked effect of enhancingimmunity (increasing NK cells) even in the former patients, i.e.,patients with terminal cancer, who can live a daily life. On the otherhand, some relationship was suspected between pathological findings ofcancer and the concentrated Basidiomycetes-X extract composition. Inpatients with adenocarcinomas, such as case 1 of colon cancer(adenocarcinoma), case 3 of mucinous cystadenocarcinoma (a type ofadenocarcinoma), case 5 of pulmonary cancer (adenocarcinoma) and case 6of gastric carcinoma (adenocarcinoma), some moves were observed in NKcell parameters after treatment with the concentrated Basidiomycetes-Xextract composition. However, case 4 was likewise a case of pulmonarycancer, but was pathologically diagnosed as having squamous cellcarcinoma. In this patient, the concentrated Basidiomycetes-X extractcomposition exerted no influence on any of the NK dynamic parameters.

Test Example 6 Course of Immunocompetence Parameters in Cancer Patients(8 Months of Treatment)

In case 3 and case 1 of Test Example 5, a mixture of 1 ml of theconcentrated Basidiomycetes-X extract composition and 1 ml of purifiedwater was orally administered 3 times daily, after each meal, insuccession to Test Example 5. The course of immunological parametersafter more than 6 months of treatment is shown in Tables 16 and 17.

(Case 3)

TABLE 16 Before After 3 After 8 treatment weeks of months of (lymphocytetreatment treatment count: LC) (LC) (LC) WBC count 3,100 3,000 2,900 RBCcount 3,420,000 3,340,000 3,430,000 Hb 11.6 11.1 10.4 Ht 34.7 33.5 32.6CD57+CD16+ (%) 5.5% 5.8% 3.1% NK activity moderate CD57+CD16− (%) 21.8%16.0% 13.4% NK activity weak CD57−CD16+ (%) 7.7% 6.8% 5.3% NK activitystrong CD3+HLA-DR+ (%) 15.5% 13.1% 31.6% Activated CD3 cells

In the present patient, moderate to strong NK activity was exhibited asterminal cancer progressed.

Lymphocytes gradually decreased. On the other hand, activatedlymphocytes were not markedly changed after 3 weeks of treatment, butincreased to 31.6% in 8 months. Thus, increases in lymphocytes(activated) similar to those after LAK (lymphokine activated killer)therapy were observed.

(Case 1)

TABLE 17 Before After 3 After 8 treatment weeks of months of (lymphocytetreatment treatment count: LC) (LC) (LC) WBC count 7,800 7,500 14,400RBC count 4,170,000 3,720,000 3,760,000 Hb 11.5 10.1 9.1 Ht 35.0 30.528.9 CD57+CD16+ (%) NK 6.6% (149)  8.0% (250) 5.0% activity moderateCD57+CD16− (%) NK 17.6% (396)  17.6% (549) 26.6% activity weakCD57−CD16+ (%) NK 4.6% (104)  4.1% (128) 2.9% activity strongCD3+HLA-DR+ (%) 9.9% (223) 10.6% (331) 22.7% Activated CD3 cells

In the present patient, CD57+CD16+ with moderate NK activity increasedafter 3 weeks of oral administration. Moreover, CD57+CD16− cells withweak NK activity increased after 8 months of oral administration. Inaddition, the activated CD3 cells increased to 10.6% at 3 weeks oftreatment, and to 22.7% after 8 months of treatment.

In conclusion, NK activity slightly increased after treatment incomparison with that before treatment.

The finding worthy of notice was that CD3+HLA−DR+ cells, markers ofactivated T lymphocytes, remarkably increased after oral administration.This outcome is normally observed after LAK therapy and, without doubt,is considered to be the extraordinary outcome of the Basidiomycetes-Xextract composition.

In patients receiving long-term treatment with the Basidiomycetes-Xextract composition, marked increases in activated lymphocytes similarto those after LAK therapy were observed, although this was the outcomein 2 patients. Based on this finding, further study seems to benecessary in an increased number of patients. However, theBasidiomycetes-X extract composition was suggested to have the potentialof increasing activated T lymphocytes and directing the immune systemtoward exclusion of cancer in patients with terminal cancer.

Example 10

Foods were cooked in accordance with the following recipes using edibleBasidiomycetes. In all foods, the organoleptic sensation of edibleBasidiomycetes was satisfactory, and its taste was good and went wellwith the foods.

1. Pasta

Just boiled pasta and sliced edible Basidiomycetes are lightly pan-friedin olive oil. Then, the mixture is preferredly seasoned with a seasoningsuch as salt or pepper. Once the edible Basidiomycetes is cookedthrough, the food is ready.

2. Pizza

Slices of raw edible Basidiomycetes are arranged on pizza dough, cheeseis sprinkled, and this combination is baked in an oven. Once cheese ismelted uniformly, the food is ready.

3. Deep-Fried Seasoned Meat or Fish

Chicken or fish is preliminarily seasoned with soy sauce or seasoningsweet sake. The pre-seasoned chicken or fish is sprinkled withErythronium japonicum starch, and slightly soaked in beaten eggs. Then,sliced raw edible Basidiomycetes is evenly pressed against the chickenor fish, and the thus treated chicken or fish is deep-fried in oil. Oncethe edible Basidiomycetes becomes crisp, the food is ready.

4. Omelet

Eggs are beaten, and seasoned in the desired manner with a seasoningsuch as salt or pepper. Finely cut raw edible Basidiomycetes is added,followed by further stirring. Then, the beaten eggs with the othermaterial are poured into a frying pan hot enough for an oil to smokelightly. The beaten eggs are agitated so as not to become solid, and theflame is turned down. While the surface of the eggs is solidified withthe remaining heat of the frying pan, the egg material is rolled. Whenit is golden brown on the surface, and half-done inside, the food isready.

INDUSTRIAL APPLICABILITY

As described above, the present invention can provide Basidiomyceteswhich is a novel mushroom having an excellent immunopotentiating action,a Basidiomycetes extract composition, health foods andimmunopotentiators using the Basidiomycetes extract composition, andedible Basidiomycetes.

Mention of Microorganism

Name of Deposition Organ: International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology

Address of Deposition Organ: Chuo Dai-6, Higashi 1-1-1, Tsukuba City,Ibaragi Prefecture, Japan (postal code 305-8566)

Date of Deposition with Deposition Organ: Feb. 27, 2003

Accession Number Assigned by Deposition Organ at Deposition: FERMBP-10011

Name of Depositor: Y. Tsuno, Representative Director, Mycology TechnoKabushiki Kaisha

Address of Depositor: Bandai 4-3-20, Niigata City, Niigata Prefecture,Japan (postal code 950-0088)

The deposited microorganism was domestically deposited on Feb. 27, 2003with International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (Accession No.: FERMP-19241), and transferred to international deposition (Accession No.:FERM BP-10011) on Apr. 15, 2004.

Other information on the features of the microorganism

Type of the microorganism: Mold

Place in taxonomy: Basidiomycetes, sclerotium (hypha mass) unidentifiedin species

Culture conditions:

-   -   Name of culture medium . . . Potato glucose agar medium    -   Composition of culture medium . . . Leachate of 200 g of        potatoes, 20 g glucose, 20 g agar per 1000 mL of the culture        medium    -   pH of culture medium . . . 5.6    -   Sterilization conditions for culture medium: 121° C., 20 minutes        in autoclave    -   Culture temperature . . . 24° C.    -   Culture period . . . 5 days    -   Requirement for oxygen . . . Aerobic    -   Culture method . . . Aerobic    -   Requirement for light . . . Unnecessary    -   Subculture conditions . . . Transfer interval 3 months, storage        temperature 5° in cool dark place        Storage conditions:    -   Storage by freeze-drying . . . Negative    -   Storage by L-drying . . . Negative    -   Storage by freezing (around −80° C.) . . . Negative    -   Storage if the above methods are unavailable . . . Storage by        subculture (transfer interval 3 months, storage temperature 5°        in cool dark place)        Spore (conidium) formation: None

1. A Basidiomycetes extract composition comprising a solvent extract ofa hyphal mass of the strain Basidiomycetes-X FERM BP-10011 wherein saidextract has medicinal properties.
 2. The Basidiomycetes extractcomposition of claim 1, wherein the solvent is hydrophilic.
 3. TheBasidiomycetes extract composition of claim 1, wherein the solvent iswater.
 4. The Basidiomycetes extract composition according to claim 2,wherein the Basidiomycetes extract composition is obtained by heatingthe extract.
 5. The Basidiomycetes extract composition according toclaim 2, wherein the Basidiomycetes extract composition is obtained bypressurization of the extract.
 6. The Basidiomycetes extract compositionaccording to claim 4, wherein the Basidiomycetes extract composition isobtained by pressurization of the extract.
 7. A health comprising theBasidiomycetes extract composition of claims 1 and foodstuff.
 8. Thehealth food according to claim 7, wherein the foodstuff is a drink, asnack, a concentrate.
 9. The Basidiomycetes extract composition of claim1 wherein the extract has a immunomodulating effect.
 10. The health foodof claim 8, wherein the food stuff is in the form of a powder, granule,tablet or capsule.